Generation of membrane proteins in polymer-based lipoparticles as flow cytometry antigens

Abstract A major obstacle in the study of membrane proteins is the challenge in generating purified targets in a stable and native conformation. Potentially destabilizing detergents are often used to extract targets out of their native membranes. One approach to overcome these destabilization issues is to present targets in a lipid bilayer environment. While numerous lipid bilayer presentation approaches such as Nanodiscs and liposomes have been used through the years, most still require a detergent purified protein intermediate. Styrene-maleic acid lipoparticles (SMALPs) have been developed to directly solubilize membranes without the use of detergents and present membrane proteins in a native lipid bilayer environment. These discoidal lipid bilayer particles have been evaluated on several membrane protein classes ( e.g. ion channels, transporters) and applications ( e.g. spectroscopy, electron microscopy). Here, cannabinoid receptor 1 (CB1), a class A G-protein coupled receptor, was incorporated into a SMALP (CB1-SMALP) and shown to retain its native fold through binding studies with a CB1-specific conformational antibody. The binding studies were conducted in yeast and mammalian display formats using fluorescence-activated cell sorting (FACS). Compared to detergent preparations, CB1-SMALP also exhibited a marked increase in thermostability. This study provides the framework for a platform to generate full-length membrane protein screening antigens for flow cytometry-based studies.